An extensive flow cytometric evaluation continues of patients with autoimmune lymphoproliferative syndrome (ALPS) and their extended family members, on the basis of characterization of the expanded double-negative T-cell and B-cell populations. Double-negative T-cells have been demonstrated to be alpha beta TcR, CD57+, HLA-DR+, and CD45RA+. This study has been extended to characterize the double-negative T-cells more completely including B220 expression and gamma-delta TcR T-cells in all ALPS patients. In addition, we have initiated expanded characterization of the B cells, directed at memory B cells using CD27 and B220 assessment in these patients. The observations in the B cells of ALPS patients are tied directly to an additional active protocol directed at the assessment of B220 expression on human lymphocytes. The relative deficiency in CD4/CD25 T cells that we have identified has resulted in the initiation of functional studies directed at this T cell subpopulation to assess if the immunophenotypic findings represent a functional defect in immunoregulatory T cells that could explain the genotype phenotype disparity in families with ALPS type 1a. This approach has met with unanticipated problems in developing a consistent ex vivo indicator system for inhibition. As a result of these difficulties we have changed our initial approach and have developed a quantitiative RTPCR assay for Fox P3 and have validated that this distinguishes the CD25 bright positive CD4 T cells (Fox P3 positive) from those that are CD25- (Fox P3 negative). We are currently collecting mRNA from ALPS type Ia patients and family members for Fox P3 evaluation as well as developing culturing conditions that allow expansion of immunoregulatory T cells to compare the expression of these cells in controls versus ALPS patients. We have also extended the Fox P3 assay to ATL cells based on the possibility that this disease represents a leukemic expansion of immunoregulatory T cells.